The elucidation of the mechanisms that control gene expression, in eucaryotic cells, remains a fundamental problem in molecular biology. In this proposal, the developmentally regulated assembly of functional mitochondria will be studied -in the protozoan parasite Trypanosoma brucei. The trypanosome modulates it mode of ATP production as the parasite alternates between the warm-blooded host and the insect vector. Within the glucose rich bloodstream of the mammal, trypanosomes repress mitochondrial activities, lack detectable cytochromes and rely on glycolysis for ATP production. In the insect, the trypanosomes rapidly develop an active electron transport chain and ATP is produced via oxidative phosphorylation. The mechanisms regulating the expression of mitochondrial genes and nuclear genes encoding mitochondria] proteins are among the major questions to be addressed in this proposal. The synthesis, processing and stability of mitochondrial transcripts will be examined, as well as, the coordinate expression of nuclear genes encoding mitochondrial proteins. In order to better understand the process of mitochondrial assembly in trypanosomes and to examine possible regulatory processes, two unique, aspects of trypanosome mitochondrial assembly will be studied in detail. (1) The editing of mitochondrial transcripts, and (2) the import of nuclear encoded tRNAs into mitochondria. RNA editing results in the addition or deletion of uridines in mitochondrial transcripts of trypanosomes. Editing is fundamentally important in trypanosome mitochondrial assembly since it alters the reading frame of mitochondrial mRNAs and forms translation initiation codons in some mitochondrial mRNAs. Our studies will focus on the mechanism of RNA editing by establishing an in vitro editing system and also by biochemical characterization of the proteins and RNA components involved in the editing process. Once we have established the basic mechanism of RNA editing, we will examine the regulation of editing during the developmental cycle of T. brucei. Since most, if not all, T. brucei mitochondrial tRNAs are nuclear encoded and imported into the mitochondrion the tRNA import pathway represents an unusual feature of trypanosome biology. We will examine the import of these tRNA both in vivo and in vitro to determine the properties of the tRNAs which allow them to be targeted to the mitochondrion and imported. In addition, the role of mitochondrial proteins and cytosolic factors will be evaluated in the in vitro import system. These studies will provide us with a better understanding of biochemical processes involved in trypanosome mitochondrial biogenesis, RNA editing, and tRNA maturation and import.